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Neuropathic pain (NP) imposes a significant burden on individuals, manifesting as nociceptive anaphylaxis, hypersensitivity, and spontaneous pain. Previous studies have shown that traumatic stress in the nervous system can lead to excessive production of hydrogen sulfide (H2S) in the gut. As a toxic gas, it can damage the nervous system through the gut-brain axis. However, whether traumatic stress in the nervous system leading to excessive production of H2S in the gut can ultimately cause neuropathic pain through the gut-brain axis remains to be investigated. This study established a model of chronic constriction injury (CCI) in mice to determine its effects on gut H2S production, the associated damage via the gut-brain axis, the potential neuropathic pain, as well as the probable mechanism. A CCI mouse model was developed using a spinal nerve ligation approach. Subsequently, the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were used to determine the mice’s pain thresholds. A variety of assays were performed, including immunofluorescence, western blotting, real-time quantitative Polymerase Chain Reaction (PCR), and membrane clamp whole-cell recordings. Mice subjected to CCI showed decreased MWT and TWL, decreased ZO-1 staining, decreased HuD staining, increased Glial fibrillary acidic protein (GFAP) staining, increased expression of tumor necrosis factor-alpha (TNF-α) protein and interleukin-6 (IL-6) protein, increased expression of NMDAR2B (NR2B) protein and NR2B mRNA, increased colocalization of vGlut2- and c-fos-positive cells, and a higher amplitude of evoked excitatory postsynaptic potential (EPSP) compared to Sham group. These changes were significantly reversed by H2S inhibitor treatment, and the specific NMDA receptor inhibitor MK-801 effectively restored the neurotoxicity of H2S. H2S is involved in CCI-induced neuropathic pain in mice, which might be mediated by the activation of the NMDA signaling pathway. [ABSTRACT FROM AUTHOR]

Widespread cortical accumulation of misfolded pathological tau proteins (ptau) in the form of paired helical filaments is a major hallmark of Alzheimer's disease. Subcellular localization of ptau at various stages of disease progression is likely to be informative of the cellular mechanisms involving its spread. Here, we found that the density of ptau within several distinct rostral thalamic nuclei in post-mortem human tissue (n = 25 cases) increased with the disease stage, with the anterodorsal nucleus (ADn) consistently being the most affected. In the ADn, ptau-positive elements were present already in the pre-cortical (Braak 0) stage. Tau pathology preferentially affected the calretinin-expressing subpopulation of glutamatergic neurons in the ADn. At the subcellular level, we detected ptau immunoreactivity in ADn cell bodies, dendrites, and in a specialized type of presynaptic terminal that expresses vesicular glutamate transporter 2 (vGLUT2) and likely originates from the mammillary body. The ptau-containing terminals displayed signs of degeneration, including endosomal/lysosomal organelles. In contrast, corticothalamic axon terminals lacked ptau. The data demonstrate the involvement of a specific cell population in ADn at the onset of the disease. The presence of ptau in subcortical glutamatergic presynaptic terminals supports hypotheses about the transsynaptic spread of tau selectively affecting specialized axonal pathways. [ABSTRACT FROM AUTHOR]

Rationale: Chronic stress exposure disrupts the medial prefrontal cortex's (mPFC) ability to regulate impulses, leading to the loss of control over alcohol drinking in rodents, emphasizing the critical role of this forebrain area in regulating alcohol consumption. Moreover, chronic stress exposure causes lateralization of mPFC functions with volumetric and functional changes, resulting in hyperactivity in the right hemisphere and functional decrease in the left. Objectives: This study investigated the inhibitory role of the left prelimbic cortex (LPrL) on ethanol consumption induced by chronic social defeat stress (SDS) in male mice and to examine if inactivation of the LPrL causes disinhibition of the right mPFC, leading to an increase in ethanol consumption. We also investigated the role of lateralization and neurochemical alterations in the mPFC related to ethanol consumption induced by chronic SDS. To this end, we examined the activation patterns of ΔFosB, VGLUT2, and GAD67 in the left and right mPFC. Results: Temporarily blocking the LPrL or right PrL (RPrL) cortices during acute SDS did not affect male mice's voluntary ethanol consumption in male mice. When each cortex was blocked in mice previously exposed to chronic SDS, ethanol consumption also remained unaffected. However, male mice with LPrL lesions during chronic SDS showed an increase in voluntary ethanol consumption, which was associated with enhanced ΔFosB/VGLUT2-positive neurons within the RPrL cortex. Conclusions: The results suggest that the LPrL may play a role in inhibiting ethanol consumption induced by chronic SDS, while the RPrL may be involved in the disinhibition of ethanol consumption. [ABSTRACT FROM AUTHOR]

Neuropathic pain is a chronic pain caused by direct damage to the peripheral or central nervous system, characterized by hyperalgesia, allodynia, and spontaneous pain. Hydrogen sulfide (H2S) therapy has been applied for neuropathic pain treatment, although the underlying mechanisms remain unknown. In this study, we sought to ascertain whether H2S therapy could alleviate neuropathic pain in a model of chronic constriction injury (CCI) and, if so, the potential mechanism. A CCI model was established in mice through a spinal nerve ligation method. Intrathecal injection of NaHS was used to treat CCI model mice. The thermal paw withdrawal latency (TPWL) and mechanical paw withdrawal threshold (MPWT) were used for pain threshold evaluation in mice. A series of experiments including immunofluorescence, enzyme-linked immunosorbent assay, electrophysiological test, mitochondrial DNA (mtDNA) quantification, measurement of ATP content, demethylase activity, and western blot were performed to investigate the specific mechanism of H2S treatment in neuropathic pain. Mice with CCI exposure exhibited a decrease in MPWT and TPWL, an increase in IL-1β and TNF-α expressions, elevated eEPSP amplitude, an upregulation of mtDNA, and a reduction in ATP production, whereas H2S treatment significantly reversed these changes. Furthermore, CCI exposure induced a remarkable increase in vGlut2- and c-fos-positive as well as vGlut2- and Nrf2-positive cells, an increase in Nrf2 located in the nucleus, and an upregulation of H3K4 methylation, and H2S treatment further enhanced these changes. In addition, ML385, a selective Nrf2 inhibitor, reversed the neuroprotective effects of H2S. H2S treatment mitigates CCI-induced neuropathic pain in mice. This protective mechanism is possibly linked to the activation of the Nrf2 signaling pathway in vGlut2-positive cells. [ABSTRACT FROM AUTHOR]

The expression of glutamate decarboxylase GAD65/67, an enzyme of GABA synthesis, and vesicular glutamate transporter 2 (VGLUT2) in the arcuate, dorsomedial, and ventromedial nuclei of the hypothalamus of young (3 months), adult (12 months), and old male rats (24 months) was studied by Western blotting. In old rats, an increase in the expression of GAD65/67 in the arcuate and dorsomedial, VGLUT2 in the arcuate, dorsomedial, and ventromedial nuclei was observed. Thus, an increase in opposite processes of inhibition and excitation is observed in the hypothalamic nuclei during aging. [ABSTRACT FROM AUTHOR]

The lateral habenula (LHb), which is a critical neuroanatomical hub and a regulator of midbrain monoaminergic centers, is activated by events resulting in negative valence and contributes to the expression of both appetitive and aversive behaviors. However, whole-brain cell-type-specific monosynaptic inputs to the LHb in both sexes remain incompletely elucidated. In this study, we used viral tracing combined with in situ hybridization targeting vesicular glutamate transporter 2 (vGlut2) and glutamic acid decarboxylase 2 (Gad2) to generate a comprehensive whole-brain atlas of inputs to glutamatergic and γ-aminobutyric acid (GABA)ergic neurons in the LHb. We found >30 ipsilateral and contralateral brain regions that projected to the LHb. Of these, there were significantly more monosynaptic LHb-projecting neurons from the lateral septum, anterior hypothalamus, dorsomedial hypothalamus, and ventromedial hypothalamus in females than in males. More interestingly, we found a stronger GABAergic projection from the medial septum to the LHb in males than in females. Our results reveal a comprehensive connectivity atlas of glutamatergic and GABAergic inputs to the LHb in both sexes, which may facilitate a better understanding of sexual dimorphism in physiological and pathological brain functions. [ABSTRACT FROM AUTHOR]

To better understand GABAergic transmission at two targets of basal ganglia downstream projections, the pedunculopontine (PPN) and laterodorsal (LDT) tegmental nuclei, the anatomical localization of GABAA and GABAB receptors was investigated in both nuclei. Specifically, the total number of neurons expressing the GABAA receptor γ2 subunit (GABAAR γ2) and the GABAB receptor R2 subunit (GABAB R2) in PPN and LDT was estimated using stereological methods, and the neurochemical phenotype of cells expressing each subunit was also determined. The mean number of non-cholinergic cells expressing GABAAR γ2 was 9850 ± 1856 in the PPN and 8285 ± 962 in the LDT, whereas those expressing GABAB R2 were 7310 ± 1970 and 9170 ± 1900 in the PPN and LDT, respectively. In addition, all cholinergic neurons in both nuclei co-expressed GABAAR γ2 and 95–98% of them co-expressed GABAB R2. Triple labeling using in situ hybridization revealed that 77% of GAD67 mRNA-positive cells in the PPT and 49% in the LDT expressed GABAAR γ2, while 90% (PPN) and 65% (LDT) of Vglut2 mRNA-positive cells also expressed GABAAR γ2. In contrast, a similar proportion (~2/3) of glutamatergic and GABAergic cells co-expressed GABAB R2 in both nuclei. The heterogeneous distribution of GABAAR and GABABR among non-cholinergic cells in PPN and LDT may give rise to physiological differences within each neurochemical subpopulation. In addition, the dissimilar proportion of GABAAR γ2-expressing glutamatergic and GABAergic neurons in the PPN and LDT may contribute to some of the functional differences found between the two nuclei. [ABSTRACT FROM AUTHOR]

The A11 region plays a role in numerous physiological functions, including pain and locomotor activity, and consists of a variety of neurons including GABAergic, calbindin positive (Calb+), and dopaminergic (DA) neurons. However, the neurochemical nature of Calb+ neurons and their regulatory role in the A11 region remain largely unknown. In this study, we examined the kind of functional markers co-expressed in the Calb+ neurons using sections from 8-week-old rats. To examine a marker related to classical neurotransmitters, we performed in situ hybridization for vesicular glutamate transporter 2 (vGluT2) or glutamate decarboxylase (GAD) 65 and 67, in conjunction with Calb immunohistochemistry. We found cellular co-expression of Calb with vGluT2 or GAD65/67 throughout the A11 region. Nearly all Calb+/GAD65/67+ neurons were found in the rostral-middle aspect of the A11 region. In contrast, Calb+/vGluT2+ neurons were found predominantly in the middle-caudal aspect of the A11 region. For receptors and neuropeptides, we performed immunohistochemistry for androgen receptor (AR), estrogen receptors (ERα and ERβ), and calcitonin gene-related peptide (CGRP). We found that Calb+ neurons co-expressed AR in the rostral aspect of the A11 region in both male and female rats. However, we rarely find cellular co-expression of Calb with ERα or ERβ in this region. For CGRP, we found both Calb+ neurons with or without CGRP expression. These results demonstrate that Calb+ neurons co-express many functional markers. Calb+ neurons have a distinct distribution pattern and may play a variety of regulatory roles, depending on their location within the A11 region. [ABSTRACT FROM AUTHOR]

Synaptic remodeling during early postnatal development lies behind neuronal networks refinement and nervous system maturation. In particular, the respiratory system is immature at birth and is subjected to significant postnatal development. In this context, the excitatory/inhibitory balance dramatically changes in the respiratory-related hypoglossal nucleus (HN) during the 3 perinatal weeks. Since, development abnormalities of hypoglossal motor neurons (HMNs) are associated with sudden infant death syndrome and obstructive sleep apnea, deciphering molecular partners behind synaptic remodeling in the HN is of basic and clinical relevance. Interestingly, a transient expression of the neuronal isoform of nitric oxide (NO) synthase (NOS) occurs in HMNs at neonatal stage that disappears before postnatal day 21 (P21). NO, in turn, is a determining factor for synaptic refinement in several physiopathological conditions. Here, intracerebroventricular chronic administration (P7–P21) of the broad spectrum NOS inhibitor l-NAME (N(ω)-nitro-l-arginine methyl ester) differentially affected excitatory and inhibitory rearrangement during this neonatal interval in the rat. Whilst l-NAME led to a reduction in the number of excitatory structures, inhibitory synaptic puncta were increased at P21 in comparison to administration of the inactive stereoisomer d-NAME. Finally, l-NAME decreased levels of the phosphorylated form of myosin light chain in the nucleus, which is known to regulate the actomyosin contraction apparatus. These outcomes indicate that physiologically synthesized NO modulates excitatory/inhibitory balance during early postnatal development by acting as an anti-synaptotrophic and/or synaptotoxic factor for inhibitory synapses, and as a synaptotrophin for excitatory ones. The mechanism of action could rely on the modulation of the actomyosin contraction apparatus. [ABSTRACT FROM AUTHOR]

Several studies suggest that neurons from the lateral region of the SuM (SuML) innervating the dorsal dentate gyrus (DG) display a dual GABAergic and glutamatergic transmission and are specifically activated during paradoxical (REM) sleep (PS). The objective of the present study is to characterize the anatomical, neurochemical and electrophysiological properties of the SuML-DG projection neurons and to determine how they control DG oscillations and neuronal activation during PS and other vigilance states. For this purpose, we combine structural connectivity techniques using neurotropic viral vectors (rabies virus, AAV), neurochemical anatomy (immunohistochemistry, in situ hybridization) and imaging (light, electron and confocal microscopy) with in vitro (patch clamp) and in vivo (LFP, EEG) optogenetic and electrophysiological recordings performed in transgenic VGLUT2-cre male mice. At the cellular level, we show that the SuML-DG neurons co-release GABA and glutamate on dentate granule cells and increase the activity of a subset of DG granule cells. At the network level, we show that activation of the SuML-DG pathway increases theta power and frequency during PS as well as gamma power during PS and waking in the DG. At the behavioral level, we show that the activation of this pathway does not change animal behavior during PS, induces awakening during slow wave sleep and increases motor activity during waking. These results suggest that the SuML-DG pathway is capable of supporting the increase of theta and gamma power in the DG observed during PS and plays an important modulatory role of DG network activity during this state. [ABSTRACT FROM AUTHOR]

In both Parkinson's disease (PD) patients and MPTP-treated non-human primates, there is a profound neuronal degeneration of the intralaminar centromedian/parafascicular (CM/Pf) thalamic complex. Although this thalamic pathology has long been established in PD (and other neurodegenerative disorders), the impact of CM/Pf cell loss on the integrity of the thalamo-striatal glutamatergic system and its regulatory functions upon striatal neurons remain unknown. In the striatum, cholinergic interneurons (ChIs) are important constituents of the striatal microcircuitry and represent one of the main targets of CM/Pf-striatal projections. Using light and electron microscopy approaches, we have analyzed the potential impact of CM/Pf neuronal loss on the anatomy of the synaptic connections between thalamic terminals (vGluT2-positive) and ChIs neurons in the striatum of parkinsonian monkeys treated chronically with MPTP. The following conclusions can be drawn from our observations: (1) as reported in PD patients, and in our previous monkey study, CM/Pf neurons undergo profound degeneration in monkeys chronically treated with low doses of MPTP. (2) In the caudate (head and body) nucleus of parkinsonian monkeys, there is an increased density of ChIs. (3) Despite the robust loss of CM/Pf neurons, no significant change was found in the density of thalamostriatal (vGluT2-positive) terminals, and in the prevalence of vGluT2-positive terminals in contact with ChIs in parkinsonian monkeys. These findings provide new information about the state of thalamic innervation of the striatum in parkinsonian monkeys with CM/Pf degeneration, and bring up an additional level of intricacy to the consequences of thalamic pathology upon the functional microcircuitry of the thalamostriatal system in parkinsonism. Future studies are needed to assess the importance of CM/Pf neuronal loss, and its potential consequences on the neuroplastic changes induced in the synaptic organization of the thalamostriatal system, in the development of early cognitive impairments in PD. [ABSTRACT FROM AUTHOR]

The importance of Vesicular Glutamate Transporter 2 (VGLUT2)-mediated neurotransmission has been highlighted in studies on addiction-related phenotypes. The single nucleotide polymorphism rs2290045 in VGLUT2 has been associated with alcohol dependence, but it is unknown whether or how this association is affected by environmental factors. The present study determined whether the association of alcohol-related problems with the rs2290045 in the VGLUT2 gene was modified by negative and positive environmental factors. Three samples were included: a clinical sample of 131 adolescents followed from age 17 to 22; a general population sample of 1794 young adults; and a general population sample of 1687 adolescents followed from age 14 to 17. DNA was extracted from saliva and the rs2290045 (T/C) was genotyped. Alcohol-related problems were assessed using the Alcohol Use Disorders Identification Test. Stressful life events (SLE) and parenting were assessed by questionnaires. Gene-environment interactions were investigated using a dual statistical approach. In all samples (effect sizes 0.6–6.2%), and consistent with the differential susceptibility framework, T carriers exposed to SLE reported more alcohol-related problems if they had experienced poor parenting, and lower alcohol-related problems if they had received supportive parenting. T carriers not exposed to SLE reported higher alcohol-related problems if they had received supportive parenting and lower alcohol-related problems if they had received poor parenting. Among CC carriers, alcohol-related problems did not vary as a function of negative and positive environmental factors. In conclusion, in three samples of youths, alcohol-related problems were associated with an interaction of VGLUT2 rs2290045, SLE, and parenting. [ABSTRACT FROM AUTHOR]

Adult male mice emit highly complex ultrasonic vocalizations (USVs) in response to female conspecifics. Such USVs, thought to facilitate courtship behaviors, are routinely measured as a behavioral index in mouse models of neurodevelopmental and psychiatric disorders such as autism. While the regulation of USVs by genetic factors has been extensively characterized, the neural mechanisms that control USV production remain largely unknown. Here, we report that optogenetic activation of the medial preoptic area (mPOA) elicited the production of USVs that were acoustically similar to courtship USVs in adult mice. Moreover, mPOA vesicular GABA transporter-positive (Vgat +) neurons were more effective at driving USV production than vesicular glutamate transporter 2-positive neurons. Furthermore, ablation of mPOA Vgat+ neurons resulted in altered spectral features and syllable usage of USVs in targeted males. Together, these results demonstrate that the mPOA plays a crucial role in modulating courtship USVs and this may serve as an entry point for future dissection of the neural circuitry underlying USV production. [ABSTRACT FROM AUTHOR]

Although exposure to arsenic (As) induces developmental neurotoxicity, there is a lack of data regarding its specific effects on glutamatergic neurotransmission in offspring from dams exposed to As during gestation and lactation. In this study, the body weight, glutamate content, and expression of vesicular glutamate transporter 2 (VGLUT2) and metabotropic glutamate receptors mGluR2 and mGluR3 was examined in the striatum of offspring following treatment of the dams with As (10 or 100 mg/L NaAsO2 in drinking water). At postnatal day 21, body weight was decreased significantly, whereas the glutamate content in the striatum of offspring in the 100-mg/L As group were not significantly different from those in the control group. Although mGluR3 expression was not significantly different, VGLUT2 and mGluR2 expression was significantly lower in the striatum of offspring of As-exposed dams. These data indicate that altered glutamatergic neurotransmission may contribute to As-induced developmental neurotoxic effects. [ABSTRACT FROM AUTHOR]

Cerebellar participation in timing and sensory-motor sequences has been supported by several experimental and clinical studies. A relevant role of the cerebellum in timing of conditioned responses in the range of milliseconds has been demonstrated, but less is known regarding the role of the cerebellum in supra-second timing of operant responses. A dissociated role of the cerebellum and striatum in timing in the millisecond and second range had been reported, respectively. The climbing fibre-Purkinje cell synapse is crucial in timing models; thus, the aberrant connection between these cellular elements is a suitable model for evaluating the contribution of the cerebellum in timing in the supra-second range. The aberrant connection between climbing fibres and Purkinje cells was induced by administration of the antagonist of NMDA receptors MK-801 to Sprague-Dawley rats at postnatal days 7-14. The timing of an operant response with two fixed intervals (5 and 8 s) and egocentric sequential learning was evaluated in 60-day-old adult rats. The aberrant connections caused a reduced accuracy in the timing of the instrumental response that was more evident in the 8-s interval and a reduced number of successive correct responses (responses emitted in the correct second without any other response between them) in the 8-s interval. In addition, an inability to incorporate new information in a sequence previously learned in egocentric-based sequence learning was apparent in rats with aberrant CF-PC synapses. These results support a relevant role for the cerebellum in the fine-tuning of the timing of operant responses in the supra-second range. [ABSTRACT FROM AUTHOR]

Neuronal calcium-binding protein 1 and -2 (NECAB1/2) localize to multiple excitatory neuron populations in the mouse spinal cord. Here, we analyzed rat and human spinal cord, combining in situ hybridization and immunohistochemistry, complementing newly collated data on mouse spinal cord for direct comparisons. Necab1/2 mRNA transcripts showed complementary distribution in rodent's spinal cord. Multiple-labeling fluorescence histochemistry with neuronal phenotypic markers localized NECAB1 to a dense fiber plexus in the dorsal horn, to neurons mainly in superficial layers and to commissural interneurons in both rodent species. NECAB1-positive (+) motor neurons were only found in mice. NECAB1 distribution in the human spinal cord was similar with the addition of NECAB1-like immunoreactivity surrounding myelinated axons. NECAB2 was mainly present in excitatory synaptic boutons in the dorsal horn of all three species, and often in calbindin-D28k neuronal somata. Rodent ependymal cells expressed calbindin-D28k. In humans, they instead were NECAB2 and/or calretinin. Our results reveal that the association of NECAB2 to excitatory neuronal circuits in the spinal cord is evolutionarily conserved across the mammalian species investigated so far. In contrast, NECAB1 expression is more heterogeneous. Thus, our study suggests that the phenotypic segregation of NECAB1 and -2 to respective excitatory and inhibitory spinal systems can underpin functional modalities in determining the fidelity of synaptic neurotransmission and neuronal responsiveness, and might bear translational relevance to humans. [ABSTRACT FROM AUTHOR]

α-Synuclein has a crucial role in synaptic vesicle release and synaptic membrane recycling. Although its general expression pattern has been described in the cerebellum, the precise cerebellar structures where α-synuclein is localized are poorly understood. To address this question, we used α-synuclein immunohistochemistry in adult mice cerebellar sections. We found that α-synuclein labels glutamatergic but not glycinergic and GABAergic synaptic terminals in the molecular and granule cell layers. α-Synuclein was preferentially expressed in parallel and mossy fiber synaptic terminals that also express vesicular glutamate transporter 1 (VGluT1), while it was not detected in VGluT2-positive climbing fibers. α-Synuclein was particularly enriched in lobules IX and X, a region known to contain a high density of unipolar brush cells (UBCs). To elucidate whether the α-synuclein-positive mossy fibers belong to UBCs, we double-labeled cerebellar sections with antibodies to α-synuclein and UBC-type-specific markers (calretinin for type I and metabotropic glutamate receptor 1α (mGluR1α) for type II UBCs) and took advantage of organotypic cerebellar cultures (in which all mossy fibers are UBC axons) and moonwalker mice (in which almost all UBCs are ablated) and found that both type I and type II UBCs express α-synuclein. In moonwalker mutant cerebella, the α-synuclein/VGluT1 immunolabeling showed a dramatic decrease in the vestibulocerebellum that correlated with the absence of UBC. α-Synuclein appears to be an excellent marker for intrinsic mossy fibers of the VGluT1 subset in conjunction with UBCs of both subtypes. [ABSTRACT FROM AUTHOR]

The expression of a vesicular glutamate transporter (VGLUT) suffices to assign a glutamatergic phenotype to neurons and other secretory cells. For example, intestinal L cells express VGLUT2 and secrete glutamate along with glucagon-like peptide 1 (GLP1). We hypothesized that GLP1-positive neurons within the caudal (visceral) nucleus of the solitary tract (cNST) also are glutamatergic. To test this, the axonal projections of GLP1 and other neurons within the cNST were labeled in rats via iontophoretic delivery of anterograde tracer. Dual immunofluorescence and confocal microscopy was used to visualize tracer-, GLP1-, and VGLUT2-positive fibers within brainstem, hypothalamic, and limbic forebrain nuclei that receive input from the cNST. Electron microscopy was used to confirm GLP1 and VGLUT2 immunolabeling within the same axon varicosities, and fluorescent in situ hybridization was used to examine VGLUT2 mRNA expression by GLP1-positive neurons. Most anterograde tracer-labeled fibers displayed VGLUT2-positive varicosities, providing new evidence that ascending axonal projections from the cNST are primarily glutamatergic. Virtually all GLP1-positive varicosities also were VGLUT2-positive. Electron microscopy confirmed the colocalization of GLP1 and VGLUT2 immunolabeling in axon terminals that formed asymmetric (excitatory-type) synapses with unlabeled dendrites in the hypothalamus. Finally, in situ hybridization confirmed that GLP1-positive cNST neurons express VGLUT2 mRNA. Thus, hindbrain GLP1 neurons in rats are equipped to store glutamate in synaptic vesicles, and likely co-release both glutamate and GLP1 from axon varicosities and terminals in the hypothalamus and other brain regions. [ABSTRACT FROM AUTHOR]

In mesial temporal lobe epilepsy (MTLE), spontaneous seizures likely originate from a multi-structural epileptogenic zone, including several regions of the limbic system connected to the hippocampal formation. In this study, we investigate the structural connectivity between the supramammillary nucleus (SuM) and the dentate gyrus (DG) in the model of MTLE induced by pilocarpine in the rat. This hypothalamic nucleus, which provides major extracortical projections to the hippocampal formation, plays a key role in the regulation of several hippocampus-dependent activities, including theta rhythms, memory function and emotional behavior, such as stress and anxiety, functions that are known to be altered in MTLE. Our findings demonstrate a marked reorganization of DG afferents originating from the SuM in pilocarpine-treated rats. This reorganization, which starts during the latent period, is massive when animals become epileptic and continue to evolve during epilepsy. It is characterized by an aberrant distribution and an increased number of axon terminals from neurons of both lateral and medial regions of the SuM, invading the entire inner molecular layer of the DG. This reorganization, which reflects an axon terminal sprouting from SuM neurons, could contribute to trigger spontaneous seizures within an altered hippocampal intrinsic circuitry. [ABSTRACT FROM AUTHOR]

The A10 region contains different neurons: dopamine (expressing tyrosine hydroxylase; TH), GABA, glutamate- only (expressing the vesicular glutamate transporter 2; VGluT2), and TH- VGluT2 (coexpressing TH and VGluT2). We used three methods to investigate proteins necessary for the synthesis (aromatic l-amino acid decarboxylase, AADC) or transport (vesicular monoamine transporter; VMAT2 or dopamine transporter; DAT) of dopamine within TH neurons in the A10 region. By in situ hybridization-immunohistochemistry, we found that all TH neurons in the A10 region had AADC, but not all had VMAT2, DAT or D2 receptors (DR). To determine whether TH- VGluT2 neurons account for TH neurons lacking these dopamine markers, we implemented an anatomical 'mirror technique', and found that not all TH- VGluT2 neurons lacked VMAT2, DAT or DR. Next, by quantitative RT-PCR of individual micro-dissected TH neurons, we discovered two classes of TH- VGluT2 and three classes of TH- only neurons with different latero-medial distribution. Some of the TH- VGluT2 neurons had both VMAT2 and DAT ( TH- VGluT2 Class 1); others lacked detectable levels of both transporters ( TH- VGluT2 Class 2). Most of the TH- only neurons contained VMAT2 and DAT ( TH- only Class 1), a few had DAT without detectable VMAT2 ( TH- only Class 2), and others lacked detectable levels of both transporters ( TH- only Class 3). We concluded that (a) the majority of TH neurons lacking DAT are TH- VGluT2 neurons, (b) very few TH- only neurons express DAT without VMAT2, and (c) TH- VGluT2 neurons lacking DAT also lack VMAT2. Thus, the A10 region contains dopamine neurons with differential compartmentalization and unique signaling properties. [ABSTRACT FROM AUTHOR]

We studied the organization and spinal projection of the mouse red nucleus with a range of techniques (Nissl stain, immunofluorescence, retrograde tracer injections into the spinal cord, anterograde tracer injections into the red nucleus, and in situ hybridization) and counted the number of neurons in the red nucleus (3,200.9 ± 230.8). We found that the rubrospinal neurons were mainly located in the parvicellular region of the red nucleus, more lateral in the rostral part and more medial in the caudal part. Labeled neurons were least common in the rostral and caudal most parts of the red nucleus. Neurons projecting to the cervical cord were predominantly dorsomedially placed and neurons projecting to the lumbar cord were predominantly ventrolaterally placed. Immunofluorescence staining with SMI-32 antibody showed that ~60% of SMI-32-positive neurons were cervical cord-projecting neurons and 24% were lumbar cord-projecting neurons. SMI-32-positive neurons were mainly located in the caudomedial part of the red nucleus. A study of vGluT2 expression showed that the number and location of glutamatergic neurons matched with those of the rubrospinal neurons. In the anterograde tracing experiments, rubrospinal fibers travelled in the dorsal portion of the lateral funiculus, between the lateral spinal nucleus and the calretinin-positive fibers of the lateral funiculus. Rubrospinal fibers terminated in contralateral laminae 5, 6, and the dorsal part of lamina 7 at all spinal cord levels. A few fibers could be seen next to the neurons in the dorsolateral part of lamina 9 at levels of C8-T1 (hand motor neurons) and L5-L6 (foot motor neurons), which is consistent with a view that rubrospinal fibers may play a role in distal limb movement in rodents. [ABSTRACT FROM AUTHOR]

It is not known whether a glutamate signaling system is involved in muscle inflammation (myositis). In the present study, we examined this question in the soleus muscle in a laboratory model of myositis resulting from repetitive overuse induced by electrical stimulation and injection of pro-inflammatory substances. Sections of rabbit soleus muscle with an induced myositis, i.e., exhibiting infiltration of inflammatory cells, were examined immunohistochemically using antibodies against vesicular glutamate transporter VGluT2 and the glutamate receptor NMDAR1. In situ hybridization for demonstration of VGluT2 mRNA was also performed. Specific reactions for both VGluT2 and NMDAR1 could be observed immunohistochemically in the same cells. In situ hybridization demonstrated the occurrence of VGluT2 mRNA in the cells. Double staining showed that the VGluT2 reactions were detectable in cells marked with T cell/neutrophil marker and in cells expressing eosinophil peroxidase. These data suggest the occurrence of previously unknown glutamate-mediated autocrine/paracrine effects within the inflammatory infiltrates during the development of muscle inflammation. [ABSTRACT FROM AUTHOR]

Purpose: The glutamatergic projection from the cerebral cortex and the thalamus extensively innervates the neostriatal neurons. However, some conflicts in the published literatures about cortical and thalamic intrastriatal synaptic terminals still need to be resolved. The present study intends to further elucidate the morphological characteristics of these two types of the terminals and their neurons. Methods: The corticostriatal and thalamostriatal terminals were immunolabeled for vesicular glutamate transporter type 1 (VGluT1) and 2 (VGluT2), respectively, and their neurons were retrograde labeled by biotinylated dextran amine 3,000 molecular weight (BDA3k) injection into the dorsolateral striatum of rats. The characteristics of the corticostriatal and thalamostriatal terminals were observed at the LM and EM levels, and the data were statistically analyzed with SPSS10.0 software. Results: We observed that 63.53% of VGluT1+ terminals synapsed on dendritic spines, which was different from VGluT2+ terminals with the equal percentage of synapses on spines and dendrites (14.88 and 17.86%, respectively). Notably, VGluT1+ axospinous synaptic terminals were remarkably larger than VGluT2+ axospinous synaptic terminals. Terminal size-frequency distribution analysis showed that VGluT1+ terminals were within the size ranges of 0.4-0.5 and 0.8-0.9 μm, and VGluT2+ terminals were in the ranges of 0.4-0.5 and 0.6-0.7 μm. Perforated-postsynaptic densities (-PSDs) were more frequently found in VGluT1+ axospinous synaptic terminals than in VGluT2+ axospinous terminals. Furthermore, BDA3k-labeled corticostrital neurons were larger in perikaryal diameter than the thalamostriatal neurons, and they were also categorized as the two main populations based on their size-frequency distribution. Conclusions: The morphological characteristics of corticostriatal and thalamostriatal terminals and neurons have implications for understanding the roles of synaptic plasticity in adaptive motor control by the basal ganglia, and they have facilitations for understanding the complexities of basal ganglia function. [ABSTRACT FROM AUTHOR]

The retinal ganglion cell layer of the eye comprises a subtype of cells characterized by their intrinsic photosensitivity and expression of melanopsin (ipRGCs). These cells regulate a variety of non-image-forming (NIF) functions such as light entrainment of circadian rhythms, acute suppression of locomotor activity (masking), and pupillary light reflex. Two neurotransmitters have been identified in ipRGCs, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP). To date, little is known about their release and interplay. Here, we describe the presence and co-localization of vesicular glutamate transporter 2 (VGLUT2; a marker of glutamate signaling) and PACAP in ipRGCs and their projections in the brain. Nine adult male Wistar rats were assigned to one of three groups; anterograde tracing ( n = 3), eye enucleation ( n = 3), and untreated ( n = 3). Under anaesthesia, rats were transcardially perfusion-fixated, after which the brains and eyes were removed for double immunohistochemical staining using a polyclonal anti-VGLUT2 antibody and a mouse monoclonal anti-PACAP antibody. Results revealed that VGLUT2- and PACAP-immunoreactivity (-ir) were present in ipRGCs and co-localized in their projections in the suprachiasmatic nucleus, the intergeniculate leaflet, and the olivary pretectal nucleus. We conclude that there is evidence to support the use of glutamate and PACAP as neurotransmitters in NIF photoperception by rat ipRGCs, and that these neurotransmitters are co-stored and probably released from the same nerve terminals. Furthermore, we conclude that VGLUT2 is the preferred subtype of vesicular transporter used by these cells. [ABSTRACT FROM AUTHOR]

Intraganglionic laminar endings (IGLEs) represent the only vagal mechanosensory terminals in the tunica muscularis of the esophagus and may be involved in local reflex control. We recently detected extensive though not complete colocalization of the vesicular glutamate transporter 2 (VGLUT2) with markers for IGLEs. To elucidate this colocalization mismatch, this study aimed at identifying markers for nitrergic, cholinergic, peptidergic, and adrenergic neurons and glial cells, which may colocalize with VGLUT2 outside of IGLEs. Confocal imaging revealed, besides substantial colocalization of VGLUT2 and substance P (SP), no other significant colocalizations of VGLUT2 and immunoreactivity for any of these markers within the same varicosities. However, we found close contacts of VGLUT2-positive structures to vesicular acetylcholine transporter, choline acetyltransferase, neuronal nitric oxide synthase, galanin, neuropeptide Y, and vasoactive intestinal peptide immunoreactive cell bodies and varicosities, as well as to glial cells. Neuronal perikarya were never positive for VGLUT2. Thus, VGLUT2 was almost exclusively found in IGLEs and may serve as a specific marker for them. In addition, many IGLEs also contained SP. The close contacts established by IGLEs to myenteric cell bodies, dendrites, and varicose fibers suggest that IGLEs modulate various types of enteric neurons and vice versa. [ABSTRACT FROM AUTHOR]